The invention pertains to a method for the production of bead dextran materials for gel chromatography and more particularly, to a method for preparing dextran materials where the crosslink density is controlled by the concentration of dextran within the mixture.
Several types of materials used in the field of gel chromatography. Materials based on dextran assume an important position among them and commercially supplied and have been commonly used already for more than 15 years. These materials consist of particles of a spherical shape and are supplied in a series, the members of which have defined properties of gradually varied quantity. A property of fundamental importance is, the crosslink density, which affects the size of pores and is manifested as the so called exclusion limit, i.e. the molecular mass of compounds which are unable to penetrate inside the particles. Swelling capacity, which is also related to crosslink density, is defined either as the water regain in ml of water in a centrifuged sample corresponding to 1 g of dry substance or as the bed volume, i.e. the volume occupied by 1 g of dry sample after swelling to equilibrium. The properties for commonly used and supplied gels covering a broad range of molecular masses are 700 to 200,000 for the exclusion limits, and in the range of 1 to 20 ml/g for the water regain.
Another important property of these materials is their chemical purity, in absence of ionic groups. For example, carboxylic groups will cause undesirable sorptions and thus reduce the efficiency of the separation in the gel chromatography.
The dextran gels are produced by methods well known in the art and protected by patents. The basic patent, Ger. (FRG) No. 1,292,883 (1969), application in Sweden 1958, describes application of crosslinked dextran gels formed in the reaction with 1-chloro-2,3-epoxypropane in bulk. The final gel is crushed, washed and classified on screens to obtain the required particle size consisting of particles with an irregular shape. According to the following patent, Brit. No. 974,054 (1964), application in Sweden 1961, bead dextran gels are prepared in a suspension process where particles of perfect spherical shape are formed. In a typical experiment, dextran is dissolved in water, a solution of sodium hydroxide is added and the mixture is dispersed in a suitable water-immiscible liquid, preferably toluene or dichloroethane, in the presence of a stabilizer. The particles are crosslinked by heating in the presence of a crosslinking agent and then the formed gel is isolated, washed, deswelled and dried. According to another analogous procedure, (D. Cvetkovic, B. Pekic, S. Jovanovic: Hem. Ind. 33 (1979), 139), mineral oil is used as a dispersion medium. A further procedure (Brit. Pat. No. 1,087,964--1967) employs as a dispersion medium the excessive 1-chloro-2,3-epoxypropane, which acts as a cross-linking agent at the same time.
However, the above mentioned known procedures have certain disadvantages. The use of mineral oil cause difficulties in washing and purification of the product. In crosslinking with an excess of 1-chloro-2,3-epoxypropane, it is necessary to reduce the one-sided reaction giving rise to pendant glycidyl and glyceroyl groups. In addition, dextran used as the starting material regularly contains lactones in the amount of about 20 to 50 micromole per gram of dry substance. These lactones are hydrolyzed in an alkaline medium during preparation of the gel so that the resulting gel contains the corresponding amount of free carboxyls.
Another disadvantage of the described procedures is the difficulty in attaining the required crosslink density. It appears that in samples prepared under other constant conditions that the dependence of water regain on the ratio of reactants, i.e. of the amount of crosslinking agent to the amount of dextran, has a characteristic parabolic course. The data for dextran at the initial concentration of dextran 40%, given in the following table, may serve as an example.
______________________________________ 1-chloro-2,3-epoxypropane 7.4 8.0 8.6 12.0 17.7 25.1 g per 100 g dextran water regain 13.3 10.9 8.7 5.6 3.6 3.3 ml/g of dry substance ______________________________________
It follows from the data that the said dependence is very steep in the region of low crosslinked gels (water regain 10 to 20 ml/g). Consequently, control of the suspension process is very sensitive to small incidental variations in the weight of charges, in keeping the temperature and stirring regime, and also in the quality of the starting material. Irreproducible results often occur in the crosslink density of the products. A batch is often spoiled, for example, by incomplete crosslinking after a standard reaction time, and the raw material is then depreciated in further processing.
Preparation according to the known processes is also connected with difficulties at the other side of the practical range of water regain, 1 to 1.5 ml/g. A large excess of 1-chloro-2,3-epoxypropane is not sufficient as such and it is necessary to work with highly concentrated dextran solutions, which causes technical obstacles. It is far from easy to prepare a solution of dextran with the concentration of 60 wt.-%. This requires special kneader equipment, dissolution and homogenization of the solution takes rather a long time, the energy consumption increases, and the quality of raw material can be lowered. The concentrated dextran solutions have a high viscosity which brings about further problem in their dispersion.
Surprisingly, it has been found that the above shortcomings can be overcome relatively easily by the method of this invention.